关键词: 银屑病, 黄连解毒汤, 犀角地黄汤, 成纤维细胞, HaCaT细胞, β连环素, 疾病模型, 动物

Abstract: 【Abstract】 Objective To explore the mechanisms of action of Huanglian Jiedu decoction combined with Xijiao Dihuang decoction （HLJDT-XJDH） in regulating fibroblasts in the treatment of psoriasis. Methods A mouse model of psoriasis was established by topical application of imiquimod 5% cream on the shaved back; HLJDT-XJDH at different doses of 7.7 and 30.6 g/kg was administered via gavage for intervention, and methotrexate （2 mg/kg） served as a positive control; after 7 days, the severity of skin lesions was assessed using the psoriasis area and severity index （PASI）, while histopathological changes of skin tissues were evaluated using hematoxylin-eosin （HE） staining and Baker scoring. For in vitro experiments, fibroblasts were divided into a control group, a model group, a low-dose （5% drug-containing serum） intervention group, and a high-dose （20% drug-containing serum） intervention group; cells in the control group were cultured with 20% normal rat serum for 24 hours; in the model group, cells cultured with 20% normal rat serum were stimulated with 5 ng/ml tumor necrosis factor （TNF）-α and 50 ng/ml interleukin （IL）-17A for 24 hours to mimic fibroblasts during the occurrence of psoriasis; cells in the low- and high-dose intervention groups received the same stimulation as the model group, and were cultured for 24 hours with 5% and 20% HLJDT-XJDH-containing serum, respectively, but not with the 20% normal rat serum. After the above treatment, these cells were co-cultured with keratinocytes （HaCaT cells） using a Transwell system. In addition, on the basis of the control group, fibroblasts were divided into the model group, 20% drug-containing serum intervention group, and 20% drug-containing serum intervention + OE-SFRP2 group; TNF-α and IL-17A were used to stimulate the cells to simulate the psoriatic state; the treatment in the 20% drug-containing serum intervention group was carried out as previously described; in the 20% drug-containing serum intervention + OE-SFRP2 group, cells were transfected with the vector for 48 hours to establish an overexpression model, followed by culture with 20% drug-containing serum for 24 hours, without co-culture with HaCaT cells.. Cell counting kit-8 （CCK-8） assay was performed to assess cell viability, flow cytometry to measure apoptosis rates, enzyme-linked immunosorbent assay （ELISA） to detect levels of inflammatory cytokines （TNF-α, IL-1β, IL-6） as well as chemokine ligand （CXCL） 1 and CXCL12 in mouse serum or cell culture supernatant, qPCR to determine the mRNA expression of inflammatory cytokines, chemokines, cell cycle- and proliferation-related factors, as well as SFRP2 in mouse skin tissues or cells, and Western blot analysis to determine the protein expression of SFRP2, Wnt3a, and β-catenin in fibroblasts. One-way analysis of variance was employed for intergroup comparisons, and post-hoc analysis was conducted using Tukey′s test. Results In vivo mouse experiments showed that compared with the normal control group, the model group exhibited typical psoriatic characteristics in skin morphology, including significant inflammatory infiltration in skin tissues and marked epidermal thickening; compared with the normal control group, the serum levels of TNF-α （531.16 ± 28.27 pg/ml vs. 239.58 ± 10.39 pg/ml）, IL-1β （111.40 ± 5.16 pg/ml vs. 80.35 ± 3.87 pg/ml）, and IL-6 （109.17 ± 4.84 pg/ml vs. 71.73 ± 2.04 pg/ml） significantly increased in the model group, along with their mRNA expression levels in mouse skin tissues （all P < 0.001）; compared with the model group, the treatment group showed alleviated psoriatic manifestations, and significant reductions in the levels of inflammatory factors TNF-α （low-dose, high-dose, and positive control groups: 420.80 ± 29.30 pg/ml, 322.33 ± 9.40 pg/ml, 322.97 ± 12.16 pg/ml, respectively）, IL-1β （98.69 ± 4.49 pg/ml, 89.02 ± 1.56 pg/ml, 88.88 ± 2.08 pg/ml, respectively）, and IL-6 （94.07 ± 3.76 pg/ml, 80.54 ± 3.30 pg/ml, 83.21 ± 3.18 pg/ml, respectively）, as well as in their mRNA expression levels （all P < 0.001）. In in vitro fibroblast experiments, compared with the control group, the model group exhibited a significant elevation in the supernatant levels of IL-1β （126.42 ± 3.56 pg/ml vs. 34.81 ± 0.44 pg/ml）, IL-6 （459.44 ± 9.35 pg/ml vs. 115.51 ± 7.26 pg/ml）, CXCL1 （2 434.88 ± 127.63 pg/ml vs. 762.85 ± 30.60 pg/ml） and CXCL12 （3 542.14 ± 35.86 pg/ml vs. 2 095.86 ± 45.12 pg/ml）, the expression levels of their mRNAs （all P < 0.001）, as well as the protein expression levels of SFRP2, Wnt3a, and β-catenin; after intervention with HLJDT-XJDH-containing serum, all the above indices significantly decreased （all P < 0.001）. However, when 20% drug-containing serum intervention was administered simultaneously, the expression of inflammatory factors and chemokines in fibroblasts was significantly higher in the SFRP2 overexpression group than in the non-overexpression group （all P < 0.01）. When fibroblasts were co-cultured with HaCaT cells, the model group showed significantly increased cell viability but a decreased apoptosis rate of HaCaT cells compared with the control group, while the low- and high-dose intervention groups showed significantly decreased cell viability but increased apoptosis rates of HaCaT cells compared with the model group （all P < 0.05）. Conclusion HLJDT-XJDH may exert therapeutic effects in psoriasis by downregulating the SFRP2/Wnt/β-catenin signaling pathway, thereby inhibiting fibroblast activation and inflammatory process, which subsequently suppresses the proliferation of keratinocytes and the activation of inflammatory cells.

Key words: Psoriasis, Huang Lian Jie Du Tang, Xi Jiao Di Huang Tang, Fibroblasts, HaCaT cells, beta Catenin, Disease models, animal